Abstract Arthropod-borne viruses (arboviruses) are undoubtedly a major public health concern and a cause of significant morbidity and mortality worldwide. Among the arboviruses, mosquito-borne viruses collectively constitute some of the most important human pathogens. Recently, we have observed significant expansions in their geographic distribution, substantial increases in their virulence, and their adaptation to new vectors that have now increased transmission rates to humans. The importance of these viruses is further underscored by the lack of available vaccines, therapeutics, or reliable measures that can be implemented to diminish disease. Recently, arbovirologists have become increasingly interested in the discovery and characterization of novel insect-specific viruses, in particular, mosquito-specific flaviviruses. Unlike the much better known mosquito- and tick-borne flaviviruses, which alternately infect vertebrate and invertebrate hosts, mosquito-specific flaviviruses appear to replicate only in mosquitoes and mosquito cells. A large number of insect-specific flaviviruses has been isolated from and/or detected in mosquitoes and sandflies collected worldwide, with very high prevalence rates among affected populations. Although these insect-specific flaviviruses are abundant in nature, very little is known about their ecological niche, their role in the evolutionary history of flaviviruses, the genetic determinants that render them unable to replicate in vertebrate cells, or to what extent they affect transmission of vertebrate-pathogenic flaviviruses in nature. Several studies have demonstrated that these viruses are mosquito-specific using conventional virologic techniques, but there are currently no data that describe their lack of replication in vertebrate cells at a cellular or molecular level, and no work has been attempted to determine exactly where the restriction to replication in vertebrate cells exist for these flaviviruses. Additionally, very few studies with conflicting results have been performed to determine the effect of mosquito- specific flaviviruses on their vertebrate-pathogenic flavivirus relatives, and no work has been done to identify the mechanism by which superinfection exclusion occurs among these viruses. This project will utilize a recently discovered insect-specific flavivirus (tentatively designated Aripo virus; ARPV) that is evolutionarily closely related to the vertebrate-pathogenic flaviviruses, as a prototype, to achieve the following aims: (1) to determine at what stage of the virus? replication cycle is ARPV restricted from replicating in vertebrate cells, and (2) to identify the mechanism by which ARPV precludes superinfection by vertebrate-pathogenic flaviviruses in mosquitoes. This study will utilize a wide array of techniques to determine if ARPV is restricted from replication in vertebrate cells because of an inability to attach and enter, replicate and/or translate its genome, or correctly assemble and release its progeny virions. We will also determine how ARPV affects the RNAi pathways in vitro and in vivo, and identify which components are responsible for preventing the subsequent superinfection by vertebrate-pathogenic flaviviruses, in a medically important vector species. Altogether, the results obtained from this project will be instrumental in understanding flavivirus biology, and will be readily translated to design new therapeutic targets, attenuate flaviviruses for novel vaccine approaches, and design biological control measures for important vectors through the use of mosquito-specific viruses or activation of key components in the RNAi pathway to prevent subsequent flavivirus infection. My most recent work involves the isolation and characterization of a novel insect-specific flavivirus, named Aripo virus (ARPV) that was isolated from mosquitoes collected in Trinidad. The complete ARPV genome was determined and phylogenetic analyses showed that ARPV is closely related to the pathogenic mosquito-borne flaviviruses. My work also shows that prior infection with ARPV reduces superinfection and dissemination rates of West Nile virus in vivo in Ae. aegypti mosquitoes. Experimentally, ARPV infection is also efficiently transmitted vertically in Ae. aegypti mosquitoes, at least to the F3 generation. There is currently no work being undertaken to determine exactly where the restriction to replication in vertebrate cells exist for these insect- specific flaviviruses, and no work aimed at identifying the mechanism by which superinfection exclusion occurs between insect-specific and pathogenic flaviviruses. These new avenues of research involving flavivirus host restriction and mosquito innate immunity will form the foundation of my future research as I transition into an independent position. My preliminary work shows ARPV can attach and enter, and replicate and translate its genomic material in vertebrate cells, clearly demonstrating this is a productive line of research. Extensive theoretical and practical training in new experimental techniques are required to effectively complete my aims and successfully transition to an independent faculty. UTMB offers a vast plethora of opportunities including training in lab and project management, manuscript and grant writing, and mentoring and communication. UTMB contains renowned arbovirologists, RNA biologists, faculty mentoring programs, core facilities and many intramural funding options that will effectively guide and support my transition toward independent investigator status. This award will provide financial support and protected time to generate high quality data for R grant submissions. This award will support my long-term goal of leading an effective arbovirus research team.